A key negative regulator of the p53 tumor suppressor protein is Mdm2, a protein which can both suppress p53 transcriptional activation of its target genes and induce proteasomal degradation of p53. MdmX, the Mdm2 homologue, is also critical for keeping p53 in check until it is needed. Identification of new means of preventing the suppression of p53 by both Mdm2 and MdmX is an important component of the planned studies. We will study the regulation of p53 by Mdm2 and MdmX using biochemical and cellular approaches. It is planned to examine in detail several aspects of the RING domains of these two proteins. We will test a number of hypotheses including the proposition that the C-termini of Mdm2 and MdmX regulate the activity and specificity of the Mdm2 E3 ligase towards different target proteins. Another hypothesis is that there are critical differences in the modes by which Mdm2 and MdmX repress p53 transactivation which will be tested by extensive biochemical analyses. The third hypothesis, that stress signaling to p53 involves nucleolar disruption, is relevant to our finding that ribosomal protein RPS7 is both a regulator and substrate of Mdm2 and that RPS7 impacts the activity of Mdm2 after various forms of stress. We hope to eventually exploit findings from the proposed work for novel cancer therapies. PUBLIC HEALTH RELEVANCE: The p53 tumor suppressor is circumvented in the majority of human cancers. It is therefore essential to discover how to restore its anti-oncogenic activity. The goal of the planned research is to find ways to release p53 from it negative regulators Mdm2 and MdmX in tumors.